This proposal involves an investigation of the molecular mechanism of enterotoxin B production and its control in Staphylococcus aureus. Enterotoxin are exoproteins produced by certain strains in culture media and in foods. They are known to be a major cause of food poisoning. It has been indicated that the enterotoxin B gene is chromosomal. The toxin gene is very labile, possibly a mobile genetic element, and is part of a unique and complex genetic system. The experimental approach involves the isolation of enterotoxin B gene(s) by molecular cloning E. coli and S. aureus. This consists of shotgun cloning of chromosomal restriction fragments onto a vector plasmid, transformation to a nontoxinogenic strain, and screening for the cloned toxin gene by a radioimmunoassay using anti-toxin serum. Once the toxin gene has been cloned and characterized by restriction mapping, an in vitro coupled transcription-translation system will be used to study the coding properties of the toxin gene. This system will also be used to identify the elements involved in the synthesis and regulation of enterotoxin B. The DNA sequence of the toxin gene(s) will be determined and the coding sequence for the toxin polypeptide identified. Sub-cloning experiments will be performed to determine if any regulatory sequences/gene are involved in enterotoxin B production. If a regulatory gene/polypeptide is involved, its precise role will be determined by using in vitro protein synthesis systems. The cloned toxin gene(s) will be used as a probe to localize the toxin genes in several enterotoxinogenic S. aureus strains by DNA-DNA hybridization. Preliminary studies will be carried out to determine the basis of the lability and mobility of the enterotoxin B gene and to see if the toxin gene is part of transposon system. Successful completion of these studies should reveal the molecular basis of enterotoxin B production and the molecular genetic processes underlying the public health problem of staphylococcal food poisoning.